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Thompson, J., Ben Ahmeid, A.A., Pickett, C., and Neil, D. (2011) Identification of Cost-Effective Measures to Improve Holding Conditions and Husbandry Practices for the Horseshoe Crab Limulus polyphemus. Project Report. University of Glasgow, Glasgow, UK. Copyright © 2011 University of Glasgow A copy can be downloaded for personal non-commercial research or study, without prior permission or charge

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Identification of cost-effective measures to improve

holding conditions and husbandry practices for the

horseshoe crab Limulus polyphemus

A Scientific Report by

Dr. John Thompson

Ms Asma A. Ben Ahmeid

Ms Claire Pickett

Professor Douglas M. Neil

December 2011

Contents

1. Introduction 1

2. Materials & Methods 10

3. Results 13

4. Discussion 20

5. Conclusions 22

6. Recommendations 23

Acknowledgements 23

References 24

Appendices 31

Scientific Report Improving the husbandry of the horseshoe crab

1

1. Introduction

1.1 Distribution and life history of the American Horseshoe crab, Limulus

polyphemus

The American or Atlantic Horseshoe crab (HSC), Limulus polyphemus, is one of only four

extant species of HSC worldwide. While wild populations of L. polyphemus are located on

the Atlantic coast of North America (from the Gulf of Mexico to Maine; Smith et al,

2002a), the three other species: Tachypleus tridentatus, Tachypleus gigas and

Carcinoscorpius rotundicauda, are found in the coastal waters of Asia, with distribution

extending from India to Japan as well as the Philippine Islands (Walls et al. 2002; Smith

and Berkson 2005). The largest L. polyphemus populations are located in Delaware Bay

and Chesapeake Bay (Faurby et al, 2010; Shuster, 1982). Despite the name these

chelicerate arthropods are more closely related to spiders and scorpions than crustaceans

and insects (Walls et al. 2002; Gerhart 2007). While there is some debate regarding the

exact classification of L. polyphemus within sub-phylum Chelicerata (Smith et al, 2002b),

they are currently classified as members of Class Merostomata, Subclass Xiphosura, Order

Xiphosurida, Suborder Limulina, Superfamily Limulacea and Family Limulidae (Walls et

al, 2002).

L. polyphemus are regarded as environmental generalists, being eurythermic and

euryhaline, able to tolerate temperatures ranging from -5 to 35°C and salinities between 5

and 35ppt (Nolan and Smith, 2009). The species is poikilothermic and are regularly found

buried in the sediment, which may be a behavioural adaptation to avoid lower temperatures

(Frankel, 1960). It is a theory supported by the presence of suitable coastal habitat to the

north of their range indicating that temperature is the likely limiting factor in their

distribution (Sekiguchi and Shuster, 2009).

Typically opportunistic omnivores, L. polyphemus have been shown to preferentially

predate upon bivalves and polychaetes, indeed bivalves have been demonstrated as

comprising over 80% of some animals diet (Botton, 1984; Ruppert & Barnes, 1994; Botton

et al, 2003; Tzafrir-Prag et al, 2010). Indeed captive Horseshoe crabs show a preference

toward thin shelled clams such Mulinia lateralis and Mya arenaria (Botton, 1984). Further

studies have also shown that L. polyphemus require sources of particulate organic matter

(POM), such as algae, to supplement their diets (Gaines et al, 2002; Carmichael and

Valiela, 2005), whilst Botton (1984) determined that 90% of animals sampled were found

to have vascular plant material within their digestive systems. Natural predators of L.

polyphemus include molluscs, crustaceans, fish, leopard sharks, eels, birds and sea turtles;

additionally they have also been fished by humans for both bait and haemolymph collection

(Walls et al, 2002).

Adult L. polyphemus over-winter beyond the subtidal zone in waters between 5 and 20

metres in depth (Shuster, 1982). Stimulated by the lengthening photoperiod they begin to

migrate shoreward in late spring, appearing in the intertidal zone around April (Shuster,

1985). Breeding appears to be primarily governed by the lunar cycle with activity peaking

during spring tides while declining during neap tides (Widener & Barlow, 1999). The

height of the breeding season occurs around the spring tides of May and June (Shuster,

1985). The end of breeding activity is signalled by the movement of the adult animals back

to deeper water (Shuster, 1985).


2

Females must move up onto the beach to spawn, typically accompanied by a male, who

‘hitches a ride’ by clasping her terminal spines using modified front legs (Cohen &

Brockman, 1983; Brockman & Penn, 1992; Walls et al, 2002). These breeding pairs may

remain attached to one another for weeks, returning to the beach for repeated spawning

(Brockman & Penn, 1992). It is common for such pairs to be accompanied up the shore by

other males, in an attempt to fertilise the female’s eggs before the attached male is able to

do so (Brockman and Penn, 1992). The females typically lay 5-7 clusters of green, ovoid

eggs (2-3mm in diameter) during each spawning event, with each cluster containing approx

3,650 eggs (Shuster, 1950). A female may lay around 88,000 eggs over the course of a

breeding season (Shuster, 1982). After fertilisation by the male/s the female covers the eggs

with sand to a depth of between 5 and 30 cm (Brockman, 1990). After 2-5 weeks the

majority of the eggs hatch into trilobite larvae (approx. 1cm in length), which remain in the

sand for several weeks (Rudloe, 1979; Rudloe, 1981a). However, in some instances a

proportion of eggs will remain within the sand and not hatch until the following spring

(Botton et al, 1992).

At the post-trilobite stage the larval horseshoe crabs moult into the first instar stage at

which point they cease the nocturnal swimming behaviour of the trilobite larval stage and

begin crawling across the sediment surface (Rudloe, 1981b). The first and all subsequent

instar stages resemble the adult, the only major difference in outward appearance being

telson length.

L. polyphemus can live up to 20 years and typically moult around 18 times, taking 9 to 12

years to reach maximum size (Shuster, 1985; Shuster & Sekiguchi, 2003). Moulting occurs

during the warmer summer months and becomes more time consuming and difficult as the

animals age (Shuster, 1982). Females reach maturity a year later and so generally moult at

least once more than their male counterparts, as a consequence females are significantly

larger than males at maturity (Shuster, 1955). Indeed the mean prosomal width of a mature

male is around 75 to 79% that of a mature female (Shuster, 1982).

1.2 Anatomy of Limulus polyphemus

1.2.1 External anatomy

L. polyphemus possess a rigid, external exoskeleton primarily composed of the

polysaccharide, chitin (Ruppert & Barnes, 1994). The animals are dorsoventrally flattened

with a carapace divided into three sections (Figure 1): the prosoma, opisthosoma and tail-

like telson (Smith and Berkson, 2005). The opisthosoma possess distinctive, flexible,

terminal spines, which the male grasps during breeding (Cohen and Brockman, 1983;

Brockman and Penn, 1992; Walls et al, 2002). While the telson is typically utilised by L.

polyphemus to right the animal when on its back (Ruppert & Barnes, 1994; Smith and

Berkson, 2005).

L. polyphemus possess 14 pairs of ventral appendages (Figure 1) (Ruppert & Barnes, 1994).

These comprise a pair of modified chelicerae, six pairs of segmented pedipalps (the first

four pairs of which are chelated), a pair of chilaria and six pairs of branchial appendages

(the first pair of which have fused to form the operculum and the remained five adapted to

form book gills) (Ruppert & Barnes, 1994; Smith and Berkson, 2005). In males the first


3

pair of pedipalps are modified and are commonly referred as ‘boxing gloves’ (Figure 2).

These are utilised to clasp the female during mating; an adaptation that allows easy

differentiation between the sexes (Walls and Berkson, 2000; Brockman and Penn, 1992).

The fifth pair of pedipalps are not involved in locomotion, but bear a lateral, spatulate

process known as a flabellum, which is utilised to direct water currents into the respiratory

chamber of the opisthosoma (Ruppert & Barnes, 1994). The fifth pair of walking legs (sixth

pair of pedipalps) are not chelated, but instead possess four leaf-like processes attached to

the end of the first tarsal segment; typically used for burrowing, to clear away mud and silt

(Ruppert & Barnes, 1994).

Vision is provided by a pair of laterally located compound eyes and a central ocellus, or

simple eye, located on the dorsal surface of the prosoma (Smith and Berkson, 2005). In

addition, L. polyphemus also possess a series of photoreceptors on their opisthosoma and

telson (Schuster 1982).

The mouth is centrally located on the ventral surface, at the confluence of the pedipalps

(Ruppert & Barnes, 1994). When a food item is detected by the chelicerae, the claws at the

ends of the pedipalps grasp it and pass it back to the gnathobases; spiny areas on the

median side of the coxa of the first four pairs of pedipalps (Ruppert & Barnes, 1994). The

gnathobases act as primitive teeth and jaws, macerating and transporting fragments of food

anteriorly (Ruppert & Barnes, 1994). These fragments are conveyed into the crab's mouth

with assistance from the chelicerae and chilaria. Waste is excreted through the anus located

on the ventral surface of the opisthosoma, slightly anterior to the telson.

1.2.2 Internal anatomy

L. polyphemus possess a large dorsally-located tubular heart; from which a well developed

arterial system extends and connects to an open vascular system via tissue sinuses (Schuster

1982; Ruppert & Barnes, 1994). The heart has eight pairs of ostia, each having two valves

through which oxygenated haemolymph enters the heart from the pericardial chamber

(Ruppert & Barnes, 1994). Once in the heart the haemolymph is pumped forward and

escapes through three pairs of aortae, one pair of cerebral arteries, and a single frontal

artery (Schuster 1982). Deoxygenated haemolymph from the tissues collects in two large

ventrally located, longitudinal sinuses (Ruppert & Barnes, 1994). The haemolymph then

flows from these ventral sinuses into the book gills, the beating of which not only results in

external water circulation, but also act as a pump driving blood through the lamellae

(Ruppert & Barnes, 1994). The haemolymph then returns to the pericardium. For the

purposes of bleeding, the cardiac sinus can be accessed through the arthrodial hinge located

between the prosoma and opisthosoma (Smith and Berkson, 2005).

Horseshoe crabs possess a basic digestive system comprising a mouth, which opens into a

cuticle-lined esophagus (Figure 1). This turns backwards into the proventriculus, which is

composed of a ‘crop’ and ‘gizzard’ (Figure 1). The crop can expand considerably to

accommodate ingested food. The food then passes into the gizzard, which has the thickest

muscular wall of the entire intestinal tract, where food is reduced to a pulp. Undigestible

material, such as fish bones or larger shell pieces are regurgitated. The pulverised food then

passes into the midgut, through the pyloric valve that protrudes from the end of the gizzard,

where it is digested. The hindgut is a short muscular tube that opens through the slit-like

anus.


4

Figure 2: Photograph showing the modified front pedipalp of a male American horseshoe crab

(L. polyphemus). From:http://www.flickr.com/photos/artour_a/1534830634/

L. polyphemus has a rudimentary nervous system, displaying a high degree of fusion

(Ruppert & Barnes, 1994). It comprises a ventrally situated cerebral ganglion, or ‘brain’,

linked to a ventral nerve cord possessing five smaller ganglia (Ruppert & Barnes, 1994).

Radiating from these are a pair of optic nerves, running directly to the two lateral eyes, 8

pairs of haemal nerves extending into the prosoma as well as numerous lateral nerves

branching into the opisthosoma (Ruppert & Barnes, 1994).

1.3 Haemolymph

1.3.1 Function and structure

Figure 1: Diagram of the external and internal anatomy of the American Horseshoe crab

(Limulus polyphemus). (From http://what-when-how.com/animal-life/subclass-merostomata/).


5

The two predominant components of L. polyphemus haemolymph are the copper-containing

respiratory protein haemocyanin and a single amoebocyte cellular component (Toh et al.

1991; Ruppert & Barnes, 1994). Haemocyanin fulfils the requirement for an oxygen

transporter while the primary role of the amoebocytes involves blood clotting (Ruppert &

Barnes, 1994). L. polyphemus haemocyanin contains two copper atoms and corresponds

closely to the minimum molecular weight, of approximately 74 kDa, of typical arthropod

haemocyanin (Sullivan et al. 1974). However, unlike most forms of haemocyanin and

haemoglobin, oxygen binding by L. polyphemus haemocyanin exhibits a reverse Bohr

effect (Sullivan et al. 1974).

L. polyphemus amoebocytes (Figure 3) are responsible for the animals immune response to

microbial invasion (Muta et al, 1993). The coagulation system employed by Horseshoe

crabs is unique, involving a serine protease zymogen cascade initiated by the presence of

bacterial endotoxins and various lipopolysaccharides (Muta et al, 1993). Once triggered,

clotting factors stored in granules within the amoebocytes are released into the

haemolymph via degranulation (Toh et al, 1991). The clot formed through activation of the

cascade is highly effective in immobilising invading microorganisms, thereby allowing

their neutralisation by antimicrobial substances, such as protein inhibitors, antimicrobial

peptides and lectins, also secreted by the amoebocytes (Miyata et al, 1989; Armstrong,

1991; Kouno et al, 2008). Consequently, this coagulation system is critical for both

haemostasis and the microbiological defence of L. polyphemus (Muta et al, 1993).

1.3.2 Limulus amoebocyte lysate (LAL)

It is the unique qualities of L. polyphemus haemolymph that make it extremely useful to

both the medical and pharmaceutical sectors. The high sensitivity of the L. polyphemus

serine protease zymogen cascade is utilised in detecting bacterial contamination of medical

and pharmaceutical products, such as intravenous drugs, vaccines and medical implements

(Walls et al, 2002).

The phenomenon was first noted in 1955 by Dr. Frederik Bang whilst working at the

Marine Biological Laboratory in Woods Hole, Massachusetts. Bang observed that an

injection of marine bacteria into the bloodstream of L. polyphemus resulted in massive

clotting (Bang, 1956). Through later research it was determined that the clotting was

triggered by endotoxins present on the surface of the bacteria (Levin & Bang, 1964a). The

active component of the clotting process was subsequently localised to the amoebocytes

with the reaction being reproducible in vitro utilising cell-free amoebocyte lysate (Levin &

Figure 3. L. polyphemus amoebocytes. (Scale, 1 square = 1mm2)

(Micrograph courtesy of Asma A. Ben Ahmeid)


6

Bang, 1964b, Novisky, 1984). The Limulus amoebocyte lysate, or LAL, assay, received

FDA approval for use in the testing of drugs, blood products, intravenous fluids and

disposable pharmaceutical devices, in the 1970's (Wall et al, 2002). The LAL assay has the

capacity to detect one millionth of a billionth of a gram of endotoxin in approximately 45

minutes (Mikkelsen, 1988, Wall et al, 2002).

Harvesting of Limulus amoebocyte lysate involves drawing haemolymph from the cardiac

sinus of live adult L. polyphemus using a large bore needle (Walls et al, 2002). Typically,

100-300 ml of haemolymph is extracted from each animal, the amount dependent on size,

sex and the period between bleedings (Rudloe, 1983). Aquarium studies have suggested

that a bled individual can restore its blood volume in 3-7 days; however, it may take up to 4

months for it to regain its pre-bled amoebocyte levels (Novisky, 1984).

Post collection, the haemolymph is centrifuged at low speed, resulting in separation of the

cellular component (amoebocytes) from the cell-free supernatant (Novisky, 1984). The

supernatant is discarded allowing the cellular component to be first homogenised and then

centrifuged at high speed. Centrifugation separates the cellular debris, while the required

clotting factors remain suspended in the resultant supernatant, referred to as the Limulus

amoebocyte lysate (LAL) (Novisky, 1984). For storage purposes the LAL is then frozen in

its raw, inactive form. It is converted to the active form via the addition of ions in the form

of sodium, calcium or magnesium salts before a patented solvent extraction is performed to

improve the reagent’s sensitivity prior to freeze-drying for long-term stability (Novitsky,

1984; Wall et al, 2002).

There are several methodologies utilised for the performance of LAL sterility tests,

however, only the three most widely used are described below. All three share the same

initial step involving the mixing, in a glass dish, of the reagent and test solution in equal

proportions (Wall et al, 2002). The first method then incubates the mixture at 37oC for

approximately 1 hour before examining for evidence of clot formation, which would

indicate the presence of endotoxin or pyrogen (Novitsky, 1984). The second method

involves assessment of the rate of turbidity of the mixture prior to the formation of a clot

(Wall et al, 2002). The final method, known as the chromogenic substrate method, involves

a colour change (yellowing) of the reagent post mixing with the degree of colouration after

a fixed period used to indicate the presence of endotoxin or pyrogen (Wall et al, 2002).

Since its discovery the LAL assay has been a significant and widely utilised tool in the

pharmaceutical industry, providing a rapid and cost effective alternative to the use of live

animals and animal products in testing for bacterial contamination (Mikkelsen, 1988; Walls

et al, 2002). In addition to its primary use in the testing of pharmaceutical products, new

applications for LAL continue to be found, such as the detection of bacterial contamination

in meat, fish, and dairy products, including frozen items (Wall et al, 2002).

1.3.3 Effects of bleeding-induced stress on L. polyphemus

Aquatic invertebrate health is affected by a variety of factors, of which physical or

physiological stress is one of the most significant (Evans et al. 1999). Both short and long-

term stressors can lead to alterations in respiratory function and structure as well as changes


7

to metabolism, osmotic balance, haemolymph volume and tissue pH (Paterson and

Spanogh, 1997). The cumulative effects of such stress may ultimately lead to reductions in

metabolic rate, animal growth and survival (Allender et al, 2010).

Stress has been shown to impact on crustacean haemocyanin function as well as altering

haemolymph composition, varying parameters such as pH and L-lactate concentration

(Carlsson and Gäde, 1986; Burnett, 1992). Gäde (1983) discovered that respiratory and

heart rates were suppressed in some arthropod species after exposure to a low oxygen

environment. In addition, during seasonal hypoxia increases in haemolymph L-lactate

levels have been observed (Whiteley et al, 1990). The increased L-lactate could be a result

of anaerobic glycolysis utilised to produce ATP under low oxygen conditions. (Hill et al.

1991). During periods of air exposure, horseshoe crabs experience respiratory acidosis (a

decrease in haemolymph pH) due to CO2 accumulation, resulting in a shift of the oxygen

equilibrium to the left (Burnett et al. 1988). Furthermore, it was determined that such

acidosis could lead to a disturbance of enzymatic pathways and potentially increase animal

mortality (Burnett et al. 1988).

During the typical collection and bleeding processes employed by researchers and

pharmaceutical workers, horseshoe crabs are exposed to a number of stressors, which may

include handling, air and sun exposure, temperature variations and of course blood loss. To

date the vast majority of L. polyphemus bled commercially for LAL production have been

wild capture animals. As a consequence limited work has been conducted examining stress

parameters and mortality post bleeding. However, there is evidence indicating that

commercial bleeding results in an increase in mortality of between 8% and 15% in wild

caught animals (Rudloe, 1983; Wall et al, 2000; Wall et al, 2002 and Hurton et al, 2009).

Rudloe (1983) showed that bleeding resulted in increased mortality in animals in the wild

by 10% in the first 12 months and by 11% in the second year. L. polyphemus mortality has

been observed to increase from 2.6% in unbled animals to 8% in bled individuals under

exposure to low and high impact stressors, such as air exposure and temperature variation

(Hurton et al, 2009). Consequently, elevated mortality likely results from synergistic

interactions between such stressors (Schisler et al. 2000; Hatch & Blaustein 2003).

Therefore, efficient and effective methods are required to assess the physiological state of

L. polyphemus. Such methods would work to minimise mortality by allowing any

overstressed or weakened animals to be selected out prior to bleeding.

1.4 Culture of L. polyphemus

Due to the increasing pressures on wild stocks of L. polyphemus measures are clearly

needed to prevent a significant and potentially irreversible population decline. However,

management of wild stocks is difficult due to the many different stakeholders involved.

Foremost amongst these are the biomedical/pharmaceutical industry and conservation

bodies both of whom wish to see population levels maintained, but for very different

reasons (Berkson & Shuster, 1999). It is this strong collective will, however, that has led to

the development of management strategies and to an increasing interest in aquaculture as a

means of both restocking and providing an alternate supply of animals for bleeding (Wall et

al, 2002; Tzafrir-Prag et al, 2010).

To date L. polyphemus have primarily been held in captivity for research purposes rather

than as stock for biomedical bleeding (Schreibman & Zarnoch, 2009). Schreibman and


8

Zarnoch (2009) regarded the production of mature L. polyphemus in aquaculture systems as

unfeasible due to their slow maturation rate and suggested instead that the enhancement of

wild stocks with cultured juveniles was a better strategy. However, given that they were

able to hatch and maintain a population of animals for 7+ years indicates that a biomedical

industry subsidiary should be able to breed and sustain large numbers of animals in

captivity for the purposes of blood extraction. This section does not detail the system setup

and culture conditions employed by the projects industrial partner, Marine Biotech Ltd., as

these are described in detail in the appendices.

As with the majority of commercially cultured invertebrate species one of the primary

issues affecting the aquaculture of L. polyphemus is the potential loss of stock due to

disease outbreaks. Indeed, stock loss due to disease is considered to be the primary

restriction in the development of the aquaculture sector (Defoirdta et al, 2007). The risk of

such outbreaks increases significantly in intensively mono-cultured animals kept at high

stocking densities (Kautsky et al, 2000). Other significant factors in the culture of L.

polyphemus are; diet composition, water quality and maintenance of abiotic parameters

(Smith & Berkson, 2005; Schreibman & Zarnoch, 2009; Carmichael et al, 2009). Poor

water quality and insufficient waste removal leads to overloading of metabolites and

environmental degradation, this puts the animals under stress and increases their

susceptibility to pathogens. Excessive fluctuations in abiotic factors such as dissolved

oxygen levels, salinity and temperature also increase stress and vulnerability to disease

(Smith & Berkson, 2005).

Culture systems are home to a diverse bacterial community, many of which are potential

pathogens which cannot be eliminated entirely by water treatment (Meyer, 1991; Schulze et

al, 2006). While these potential pathogens are also present in wild populations they rarely

cause mass mortality (Sommerset et al, 2005). However, this is not the case in cultured

populations where unnaturally high stocking densities coupled with other potential

stressors, such as suboptimal culture conditions conspire to produce in ideal environment

for disease (Hansen & Olafsen, 1999; Verschuere et al, 2000; Winton, 2001). Bacteria are

continually added to culture systems via feed, as well as through translocation of infected

stock, a problem particularly common in mollusc culture (Paillard et al, 2004).

There is relatively little information available regarding the bacterial diseases of L.

polyphemus, largely due to the fact that, to date, all commercially viable stocks were wild

and no large scale disease outbreaks of note have been observed and investigated. What is

known about the bacterial strains associated with L. polyphemus is summarised here. The

earliest recorded observation of bacterial disease in L. polyphemus occurred in 1955; where

Gram negative bacteria were isolated from the haemolymph of moribund animals (Bang,

1956). It was also observed that the haemolymph of the crabs in question did not appear to

clot and exhibited a lack of circulating amoebocytes (Bang, 1956). Since these initial

observations other potential pathogens have been isolated. Oscillatoria spp and Beggatoa

spp are causative agents in gill infections, although the latter is less invasive and less

pathogenic (Leibovitz & Lewbart, 2004; Smith & Berkson, 2005). Members of genera

Leucothrix, Vibrio, Flavobacterium, Pseudomonas and Pasturella have all been found

associated with external lesions on L. polyphemus (Smith & Berkson, 2005; Nolan &

Smith, 2009). Vibrio alginolyticus in particular is thought to be a pathogen of L.

polyphemus (Bosniak & Armstrong, 2004). However, no records of further investigation or

descriptions of the pathogenicity of these bacteria to L. polyphemus can be found.


9

As with all health issues, prevention is better than cure, therefore good animal husbandry is

vital in maintaining a healthy, productive captive L. polyphemus population (Smith &

Berkson, 2005; Schreibman & Zarnoch, 2009). This is further emphasised by the lack of

targeted antibiotic treatments specifically formulated for L. polyphemus (Smith & Berkson,

2005). Indeed for a time there was general consensus that the animal’s highly sensitive

clotting response rendered them essentially impervious to systemic bacterial infection. This

has now been discounted and the testing of L. polyphemus haemolymph for the presence of

bacteria is now advocated as a method of disease detection (Nolan & Smith, 2009).

Good aquatic animal husbandry requires regular monitoring of both animals and

environmental conditions within the culture system. Ideally, injured or diseased animals

should be isolated from the general population and examined more frequently for changes

in health status. Daily system checks should include the monitoring of water quality

parameters such as levels of; NH4, NO2, and pH, as well as abiotic factors such as dissolved

O2, temperature, salinity and redox potential. Also key is the timely removal of any solid

waste material that has accumulated within the system. Although suspended particulate

material is generally removed by filters vigilance is required to prevent the accumulation of

larger debris, particularly in holding tanks. Such matter typically takes the form of faeces

and unconsumed feed, but may also occasionally include dead animals. This waste will

increase turbidity as well as provide a growth substrate for potentially harmful bacteria and

algae. Poor water quality and insufficient waste removal leads to overloading of metabolites

and environmental degradation, putting the animals under stress and increasing their

susceptibility to pathogens (Beristain, 2005). Excessive fluctuations in abiotic factors such

as dissolved oxygen, salinity and temperature also increase stress and vulnerability to

disease (Beristain, 2005).

1.5 Project Aims

o Identification of cost-effective measures that may improve and optimise holding

conditions and husbandry practices for L. polyphemus, and thereby reduce stock

loss.

o To investigate the effect of the withdrawal of haemolymph on the physiology of L.

polyphemus and to identify any methods that may provide greater yields of

haemolymph in good condition for the extraction of the necessary biochemical

compounds (LAL).

o Investigation of the culturable bacterial flora found within the different parts of the

recirculating aquaculture system (RAS) employed by Marine Biotech Ltd. and the

identification of any potential problem areas requiring action.

o Investigation of the culturable bacterial flora associated with adult L. polyphemus

held at the Marine Biotech Limited facility, with emphasis on the identification of

potential pathogens.


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2. Materials & Methods

2.1 Physiological stress parameters

2.1.1 Experimental design

Twelve adult male L. polyphemus (average weight 1106 g; average prosomal width 219

mm) were randomly selected to examine the effects of air emersion and haemolymph

withdrawal on five physiological parameters regarded as potential indicators of stress.

These animals were divided into three experimental groups of four individuals: group one,

where haemolymph was withdrawn and the animals were immediately returned to water

and groups two and three where the animals were exposed to air (at 17oC) for a further 1

and 2 hours respectively, post bleeding. The animals were re-sampled after 2 hours to

evaluate short-term effects and again after a 2 week recovery period to evaluate any longer

term effects.

The extracted haemolymph was analysed, with measures taken of temperature, pH, protein

concentration, L-lactate level and the total number of circulating haemocytes.

2.1.2 Haemolymph extraction

After surface sterilisation with 70% ethanol solution, haemolymph was withdrawn via a

sterile 19 Gauge needle inserted through the arthroidial membrane at the base of the telson

into the cardiac sinus. The haemolymph was collected in a sterile Universal tube, samples

from which were then used for the determination of haemolymph protein concentration, L-

lactate level, temperature, and pH. For the total haemocyte counts, haemolymph was drawn

into a sterile 10 ml syringe containing 10 % neutral buffered formalin (NBF) solution (see

appendix) at a ratio of 1:2 or 1:3, blood to NBF.

2.1.3 Temperature and pH of haemolymph

Immediately upon extraction the temperature and pH of the haemolymph was determined

using a Jenway Model 3510 pH, conductivity/TDS and temperature meter (calibrated using

pH standards prior to sampling).

2.1.4 L-lactate level

Haemolymph L-lactate was measured using an Accutrend portable lactate analyser and

BM-Lactate test strips (Roche Diagnostics Ltd., West Sussex, UK) as per manufacturer’s

guidelines. The limit of detection of this analytical system was 0.8 mmol/l.

2.1.5 Haemolymph protein

Haemolymph protein concentration was determined using a standard, hand-held clinical

refractometer calibrated for serum protein (0-12g/dl). Two to three drops of fresh

haemolymph was placed on the lens of the refractometer using a disposable plastic pipette

before a reading of the protein concentration in g/dl was taken via the eyepiece.


11

2.1.6 Total haemocyte counts

Total haemocyte counts (THCs) were performed on the Haemolymph/NBF solutions using

a Neubauer haemocytometer and a Leitz Wetzlar light microscope. Haemolymph/NBF

solution (25 µl) was pipetted onto the haemocytometer, with counts performed under x40

magnification. Duplicate counts were performed for every sample.

2.1.7 Statistical analysis

Using the statistical package Minitab 16, standard statistical procedures were applied to

compare the data sets. An ANOVA test for parametric data and a Kruskal-Wallis test for

non-parametric data were used to measure differences among the three data sets. Further

comparisons between the treatment means were made using a Tukey´s range test, and a t-

test was used to compare the mean difference between two data sets. Significance was

accepted at p<0.05.

2.2 Environmental bacteriological analysis

2.2.1 Sample Collection

Water samples and biofilm swabs were collected from the recirculating, seawater,

aquaculture system at the facility of Marine Biotech Limited, Callander, Scotland.

Water samples, in triplicate, were collected from both male and female (brood stock)

holding tanks (each containing approx. 100 animals), as well as from holding tank and

protein skimmer outflow pipes. Further water samples were collected from the system’s

biofilter, each containing between 1 and 3 biofilter beads. Tank and biofilter water samples

were obtained by submerging, then opening, a sterile, 50ml Universal tube, while the

outflow pipe samples were obtained by holding an open Universal tube under the flow of

water.

Biofilm samples were collected, in triplicate, from the two animal holding tanks, biofilter

and the system sump. In addition, swabs were also taken from the surface of one of the bag

filters located within the sump. Swabs from the BBL™

CultureSwab™

Collection &

Transport system were utilised to gather biofilm from an area equivalent to 1 cm2, below

the waterline.

The surface bacterial microflora of L. polyphemus was sampled using similar methodology.

Swabs of the dorsal carapace, between the lateral eyes, and the gills of 3 males (animals 22,

33 & 67) and 3 females (animals 104, 106 & 139) were collected. The carapace samples

were gathered as per the system biofilm, whereas the gill samples were collected via the

insertion and rotation of a swab between adjacent leaves of the gill booklets. Any lesions or

wounds present on the selected animals (22 & 106) were sampled in the same fashion as the

gills. In addition, a further swab was taken from a significant carapace lesion present on a

female, number 183.

Haemolymph from the same animals was also collected for bacteriological analysis.

Haemolymph was extracted as described in Section 2.1.2 and immediately spread plated (2-

3 drops), in duplicate, onto Marine agar (Difco™

2216), Tryptic (Trypticase) Soy Agar


12

(TSA) (Oxoid™

CM0131) with 3% NaCl and Thiosulfate-Citrate-Bile salts-Sucrose

(TCBS) agar (Difco™

265020). The plates were then sealed and upon arrival at the

University of Glasgow, incubated at 15oC under aerobic growth conditions. Despite the

lack of aseptic technique during plating, all effort was made to minimise potential

contamination of the haemolymph samples.

Processing of the water and CultureSwab™

samples was undertaken, using aseptic

technique, upon return to the facilities at the University of Glasgow.

2.2.2 Sample processing

All water samples (excluding biofilter samples) underwent a ten fold serial dilution, (1ml of

sample into 9ml of sterile 3% NaCl solution; previously optimised) prior to plating. A

volume of 25µl was then pipetted and spread homogeneously onto 90mm plates of Marine

agar, TSA (with 3% NaCl) and TCBS agar. The plates were then incubated aerobically, at

15°C.

The biofilter samples were processed by transferring the plastic beads collected,

individually, to Universal tubes containing 5ml sterile 3% NaCl solution. These tubes were

then vortexed for 30 seconds in order to resuspend bacteria adhering to the bead. A 25µl

volume of each sample was then plated using the methodology previously described for the

water samples. The plates were then incubated aerobically, at 15°C.

CultureSwab™

samples were processed as follows; the tips were aseptically removed and

placed into 1.5ml Eppendorf tubes containing 500µl of sterile 3% NaCl solution. Each tube

was then vortexed for 30 seconds to resuspend any adhered bacteria. A 25µl volume of

each sample was then plated using the methodology previously described before being

incubated aerobically, at 15°C. Colony counts were performed on all plates after 24, 48 and

168 h growth time. After incubation period of 48 hours the relative proportion of Vibrio-

like strains in a given sample was then determined using the relative number of colony

forming units that developed on the TCBS agar and TSA (3% NaCl) plates. The TSA (3%

NaCl) as opposed to the Marine agar counts were selected due to the similar growth

characteristics of the TCBS and TSA media.

2.2.3 Selection and identification of isolates

After 48 h growth all visually-distinct colonies, i.e. those with differing gross colony

morphology, were picked and quadrant streaked onto fresh plates of the same media. Each

isolate was assigned an identifying number (01-41) with a record of its origin and a digital

image of its gross appearance. The plates were then incubated aerobically at 15°C and

checked after 48 and 72 hours. After sufficient growth to determine the presence of a

monoculture, single colonies were picked and continuously streaked onto fresh plates (in

duplicate) for strain identification. These plates were incubated aerobically at 15oC and

20oC, respectively, this ensured the presence of young, fast growing colonies required for

Gram staining and API®

20E™

testing. In addition, the isolates were also incubated on

slopes of appropriate media and stored at 5oC.


13

The nature of the cell wall of the isolates, i.e. Gram type, was determined via Gram staining

(Colomé et al, 1986). Identification of the isolates to species level was performed using the

API®

20E™

test system (Biomerieux, Marcy l’Etoile, France). The API®

20E™

is a

standardised identification system used to identify Enterobacteriacaea and other non-

fastidious, Gram negative bacteria. The test system was utilised as per manufacturer’s

instructions, with the exception of incubation temperature. An incubation temperature of

20oC was selected to better reflect the growth requirements of the isolates. All test strips

were initially incubated for 24 h (as per manufacturer’s instructions); however; those strips

displaying less than three positive reactions after this period were incubated for a further

24h to make allowance for the lower incubation temperature (i.e. slower growth).

Furthermore, any strips exhibiting zero positive reactions after 48 h incubation were re-run

in order to differentiate between true negative results and growth failure of the isolate.

Results for each isolate were recorded on the sheets provided and species identification

obtained (along with a percentage accuracy of the I.D.) using the Biomerieux API®

software and online database. Any identification with an accuracy of less than 50% resulted

in the isolate in question being re-tested.

3. Results

3.1 Physiological stress parameters

3.1.1 Haemolymph temperature

The bleeding procedure and associated air emersion (1 and 2 hours exposure) had no

statistically significant effect on the temperature of L. polyphemus haemolymph. Indeed the

results support the literature regarding the ectothermic nature of L. polyphemus in that the

temperature of its haemolymph reflected the prevailing ambient water and air temperatures.

3.1.2 Haemolymph pH

The mean haemolymph pH values are shown in Figure 4. Baseline (mean) haemolymph pH

was 7.5, with a statistically significant decrease in the mean of 0.3 (p= 0.010 and F= 7.83)

observed in the 1 hour post bleed samples (Figure 4; A). After the 2 week recovery period

haemolymph pH returned to a (mean) value of 7.4, determined as not significantly different

from the baseline value. Statistically significant deviation from the baseline pH value was

observed in both the 1 hour and 2 hour air-exposed groups (Figure 4; B). Mean pH dropped

to 7.0 for the 1 hour group and to 7.1 for the 2 hour exposed group (Kruskal-Wallis test: H=

11.86, p= 0.003). However, no statistically significant difference was observed in pH

between the 1 hour and 2 hour air-exposed animals.


14

Figure 4. Haemolymph pH values; A – pH values at: time of bleeding, i.e. baseline value (I), after 1

hour recovery (II) and after 2 weeks recovery (III). B – pH values at: time of bleeding i.e. baseline

value (I), after 1 hour of air exposure (II) and after 2 hours air exposure (III). Mean values ± SEM,

Baseline, n = 8; Experimental groups, n = 4.

3.1.3 L-lactate level

Of the 24 haemolymph samples analysed only five possessed L-lactate concentrations

exceeding the detection threshold of the analyser (0.8 mmol/l). The samples in question and

their L-lactate values are displayed in Table 1. Given the size of this sample set it is not

possible to draw any viable conclusions from this data regarding the effects of

bleeding/handling stress on L. polyphemus blood lactate levels, only that L. polyphemus

possesses haemolymph L-lactate levels that are lower than those of previously tested

crustacea.

Table 1. L-lactate concentration of those fresh haemolymph samples that registered on the

Accutrend portable lactate analyser. Detection threshold, 0.8 mmol/l.

Number of Haemolymph L-lactate

animals sampled concentration (mmol/l)

Baseline 2 0.9 & 0.8

1 hour air exposed 1 0.9



6.75

7.00

7.25

7.50

7.75

Initial After 1hr After 2 w eeks

pH

A I III II

6.75

7.00

7.25

7.50

7.75

Initial 1hr air exposure 2hrs air exposure

pH

B I II III


15

3.1.4 Haemolymph protein

Despite the appearance of a decline in haemolymph protein concentration with increased air

exposure, no statistically significant variation was observed between the haemolymph

protein concentrations of the baseline samples and those obtained for the experimental

groups (Table 2).

Table 2. Protein concentration of fresh haemolymph obtained from L. polyphemus – baseline

samples, after 1 hour and 2 weeks recovery and after 1 hour and 2 hours air exposure. (P & H

values as indicated; n = 4, excluding air exposed baseline where n = 8).

Baseline 6.1 ± 2.4

1 hours recovery 6.2 ± 2.5

2 weeks recovery 5.8 ± 1.2

Baseline 6.0 ± 2.3

1 hour air exposed 5.8 ± 2.5


Mean (g.100ml-1

) ± SD P-value H-value

} 0.790 } 0.48

} 0.829 } 0.38

3.1.5 Total haemocyte counts

No statistically significant differences were observed between the baseline total haemocyte

(amoebocyte) counts of any of the experimental groups (Table 3). All groups exhibited a

high degree of variance between individual animals, illustrated by the large standard

deviation values, when compared to the mean, displayed in Table 3.

Table 3. Total haemocyte (amoebocyte) counts (THC) of fresh haemolymph obtained from L.

polyphemus – baseline samples, after 1 hour and 2 weeks recovery and after 1 hour and 2 hours air

exposure. (P & H values as indicated; n = 4, excluding air exposed baseline where n = 8).

Baseline 11.8 ± 5.3

1 hours recovery 12.1 ± 5.4

2 weeks recovery 14.9 ± 6.1

Baseline 15.2 ± 6.1



} 0.105 } 4.50

} 0.309 } 1.29

Mean THC ± SD (x106) P-value H-value


16

3.1.6 Long-term effects of air exposure

Further to the air exposure data displayed in sections 3.1.1 through 3.1.5, the animals tested

were returned to the holding tank and re-sampled after a 2 week recovery period. This was

undertaken in order to ascertain whether sustained air exposure had any longer term effects

on the physiological parameters tested. L-lactate was not reassessed given the lack of

useable data collected during initial sampling.

The only test parameter to exhibit statistically significant variation over the course of the

trial as a result of air-exposure-induced stress was haemolymph pH (Table 4). In both the 1

hour and 2 hour air exposed groups blood pH displayed a statistically significant decline

(increased acidity) of 0.5 and 0.4 units respectively, with the effect being reversed in the 2

week samples (Table 4). Haemolymph temperature also displayed statistically significant

variation but only as a reflection of the ambient air/water temperature and not as a result of

air exposure per se.

Table 4. Haemolymph temperature, pH, protein concentration and total haemocyte count data for

the 1 hour air exposed and 2 hour air exposed groups after a 2 week recovery period (*); included

for comparison are the baseline and immediate post exposure values. Mean values ± SD; n = 4. (P

& H values as indicated).

Baseline

1 hour air exposed

1 hour air exposed *

P-value H-value P-value H-value P-value H-value P-value H-value

0.005 11.23 0.015 8.35 0.811 0.42 0.173 3.51

Baseline

2 hour air exposed

2 hour air exposed *

P-value H-value P-value H-value P-value H-value P-value H-value

0.001 15.75 0.03 7.36 0.887 0.12 0.328 2.23

16.1 ± 0.877.4 ± 0.08 5.3 ± 1.7

5.8 ± 2.1

7.1 ± 0.09

6.0 ± 2.1 18.3 ± 2.7

21.6 ± 6.1

7.5 ± 0.03

12.1 ± 7.8

15.5 ± 8.4

17.0 ± 1.9

7.5 ± 0.04

7.0 ± 0.05

7.4 ± 0.10

5.6 ± 2.1

5.4 ± 2.1

5.3 ± 1.6

pH Protein (g.100ml-1

) THC (x106)

18.5 ± 0.2

Temperature (oC)

18.6 ± 0.2

17.8 ± 0.3

18.2 ± 0.1

18.4 ± 0.3

17.7 ± 0.2

3.2 Environmental bacteriological analysis

3.2.1 Colony forming units and the proportion of Vibrio-like bacteria

The numbers of colony forming units (CFU) present (May 2011) at the various sample

locations within the Marine Biotech Ltd recirculating aquaculture system (RAS) are

displayed in Figure 5 (complete data tables are given in Appendix 2). For clarity, all colony

forming unit values for TSA (3% NaCl) are displayed in red, those for marine agar in blue

and where applicable, those for TCBS agar in green.


17

Figure 5. System schematic of the Marine Biotech Limited broodstock RAS, displaying average

colony forming units per 1 ml of sample. Growth on TSA (red) and marine agar (blue).

The greatest number of colony forming units within the system was observed in the water

samples obtained from the female and male holding tanks (6.41x106 & 1.23x10

6 CFU

respectively – averaged after 72 hours growth on marine agar). The highest densities grown

on TSA (3% NaCl) were observed in the protein skimmer outflow and female tank water

samples (at 3.38x105 & 3.35x10

5 CFU respectively, after 72 h). CFU values for the

haemolymph and animal surface swab samples could not be accurately determined due to

fungal contamination or low CFU density, those colonies present, however, were included

for API 20 identification.

Estimates of the relative percentage bacteria exhibiting Vibrio-like growth characteristics

are displayed in Table 5. These percentages varied considerably between samples, ranging

from 0.0% in the majority of samples, to 18.2% (protein skimmer outflow).

Table 5. Relative percentage of total CFU exhibiting Vibrio-like growth characteristics within the

samples obtained from the Marine Biotech Ltd RAS.

Female Male Female Male Tank Protein Skimmer Biofilm Beads Biofilm Bag filter Carapace Gill Blood Lesions

4.76 5.26 0.00 0.00 5.32 18.18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.88

Sump Animal

Sample location

Tank water BiofilterTank Biofilm Outflow pipes


18

3.2.2 Isolate identification

Forty one visually distinct bacterial isolates were selected from colonies arising from the

colony count plates, for identification using the Biomerieux API®

20E™

test system.

However, six of these remain unidentified as they could not be resurrected from their

original cultures. Only those colonies with differing gross colony morphology were

selected as the scope of testing was both time- and resource-limited. The successfully

identified isolates, the location from which they were sampled, along with the percentage

accuracy of the identification, are displayed in Figures 6 & 7 (full data relating to the

identification of these isolates are given in Appendix 3). All forty one isolates were Gram

negative. Ten strains were identified from the animal swabs; illustrated in Figure 6. The

remaining 25 strains were isolated from the RAS water/swab samples and can be seen in

Figure 7 on the next page.

Figure 6. Animal derived isolates with % accuracy and sample location. Isolates 35, 36, 37 & 40

were obtained from lesions at the locations indicated. [Growth media; TSA = red, TCBS agar =

green, Marine agar = blue].


19

Figure 7. Isolates derived from the Marine Biotech Limited RAS, with % accuracy and sample location.

[Growth media; TSA = red, TCBS agar = green, Marine agar = blue].

22

First Steps Award with Marine Biotech Ltd. Scientific Report

20

4. Discussion Of the physiological parameters examined, only haemolymph pH exhibited any significant

variation as a result of bleeding and air-emersion-induced stress. Numerous studies have

established that when exposed to air HSC maintain dissolved haemolymph oxygen levels of

only ca. 36% that of their normal (submerged) levels (McDonald et al. 1979; Allender et al.

2010). Given this and the fact that the animals were observed to cease gill movement upon

removal from the holding tanks, it is likely that the decline in pH is a result of respiratory

acidosis, due to the accumulation of dissolved CO2 in the haemolymph, rather than to

metabolic acidosis due to the accumulation of acidic end products (such as lactate) from

anaerobic metabolism (Defur et al, 1980, Mangum, 1986). Similar findings of respiratory

acidosis have been reported by Allender et al. (2010) following long-term (24 h) emersion

of HSCs, and by Burnett and McMahon (1987) following air exposure in certain terrestrial

crab species.

The fact that haemolymph pH can be determined both cheaply and efficiently in the field

(through the use of commercially available pH paper test strips) makes this parameter an

ideal means of assessing air emersion stress in L. polyphemus. It would be prudent to

monitor and minimise such air emersion stress in L. polyphemus, as it has been shown to

interfere with the enzymatic and osmoregulatory functions (Caillouet 1968, Prosser 1973,

Haupt et al. 2006).

The inability to accurately assess haemolymph lactate levels during these trials does not

negate the viability of this parameter as a physiological indicator of stress in L. polyphemus.

The literature indicates that L. polyphemus blood lactate concentrations are an order of

magnitude lower than those observed in crustacea (Gäde et al., 1986; Allender et al. 2010).

In addition, L. polyphemus has been shown to produce D-lactate through exposure to

environmental anoxia rather than L-lactate (Carlsson & Gäde 1986). Although these both

have the same chemical formula, they are optical isomers and can behave differently

enzymatically. However, the Accutrend analyser did provide above threshold readings for

some of the samples, indicating that the low concentration of lactate rather than optical

isomerism was responsible. Similarly low values of lactate in stressed HSC were obtained

by Allender at al. (2010) using another field instrument, a ‘point-of-care’ analyzer.

Since haemolymph lactate concentration is widely regarded as an excellent indicator of

physiological stress in crustaceans (Vermeer, 1987; Taylor & Waldron, 1997; Haupt et al.

2006; Ridgway et al. 2006) it is a parameter that certainly merits further investigation in L.

polyphemus. Consequently, it is recommended that any future study should re-examine

haemolymph lactate concentration using a more sensitive laboratory assay than provided by

the Accutrend portable lactate analyser (as in Ridgway et al. 2006).

The data support the conclusion that bled L. polyphemus appear fully recovered two weeks

post-bleeding, and can tolerate at least two hours air emersion with no apparent long-term

detrimental effects.

The greatest bacterial loading observed within the Marine Biotech Ltd recirculating

aquaculture system was exhibited by the holding tank containing female L. polyphemus.

Animal numbers in both holding tanks were approximately equal (ca. 100); however

females are approximately 20-25% larger than males (Shuster, 1982). This size difference

results in a skewing of the biomass distribution within the system, and consequently the


21

greater biomass present in the female tank may explain the higher bacterial load. A further

factor may be the difference in hormone balance between male and female, which can also

cause localised variation in bacterial populations. An improvement in overall water quality

could be achieved by reducing the system load, either by increasing the volume of the

biofilter and/or sump, or via a reduction in system biomass. A more regimented diet and

feeding regime would also aid in improving water quality. Any improvements in water

quality/ abiotic conditions would likely reduce stress considerably in the animals.

Biofilm (swab) samples contained lower than expected numbers of CFU (Costerton et al

1995) likely due to the sampling methodology employed. Given that bacteria present in the

biofilm probably have efficient mechanisms of surface attachment, any future trials will

require a more vigorous method of homogenisation to better resuspend the sampled

microorganisms.

With regard to the bacterial flora of the animals, very few CFU were recovered from the

carapace or gill samples, as is often the case in marine arthropods such as L. polyphemus.

Indeed other species of horseshoe crab (Tachypleus gigas and Carcinoscorpius

rotundicauda) have been shown to demonstrate highly effective biochemical mechanisms

to prevent bacterial biofouling (Ismail et al, 2011). As anticipated, greater numbers of

bacteria were found associated with the various lesions. Very little bacterial growth was

observed on the haemolymph plates; however, many of these plates were found to have

been contaminated by mould.

The proportion of Vibrio-like strains within the system fluctuated between 0% (majority of

locations) and 18.2% (protein skimmer outflow water). Given that Vibrio species make up a

very high proportion of the bacteria found in the marine environment (up to 80% in some

cases) (Tsukamoto et al, 1993; Lavilla-Pitogo et al, 1998) it is unusual to find such low

concentrations in a seawater RAS. This may be explained by the exclusive use of artificial

seawater at the facility, coupled with a purposefully low water temperature (sub 18oC). It is

generally accepted that Vibrio spp. are less successful at lower temperatures (Kelly, 1982;

Choi & Peters, 1992; Liu et al, 2010). For example, the optimal growth temperature for

Vibrio harveyi and Vibrio (Beneckea) vulnificus is 28°C; also V. (Beneckea) vulnificus is

rarely isolated from waters below 20°C (Kelly, 1982; Choi and Peters, 1992; Liu et al,

2010).

Of the 35 bacterial strains identified via the API® 20 E

™ identification system, only four

could potentially behave pathogenically toward L. polyphemus. These are Aeromonas

hydrophila/caviae/sobria, Aeromonas salmonicida, Ewingella americana and Vibrio

fluvialis. Two further strains were identified as the potential human pathogens,

Pseudomonas oryzihabitans and Pasteurella multocida. A full review of the species

identified from the Marine Biotech Limited facility can be found in the appendices.

Although bacterial loading within the system is not of a level to cause concern at this time

the presence of several strains, that could be potentially harmful to humans and L.

polyphemus, does merit attention. Currently, the animals housed within the system appear

well able to tolerate the presence of these strains, however, were conditions to deteriorate

these organism could become a significant problem. This issue could be addressed by the

relocation of the UV sterilisation unit from its current position to one between the biofilter

and the holding tank feeder pipe. Also the effectiveness of such UV treatment is directly

proportional to its degree of use. Consequently, its continual usage is recommended for

maximum effectiveness in the control of bacterial numbers.


22

5. Conclusions

o Haemolymph pH appears to provide an effective and practical physiological indicator

for air- exposure-induced stress in adult L. polyphemus.

o THC, haemolymph protein concentration and blood temperature exhibited no

statistically significant variation in stressed (air exposed) versus unstressed animals.

o Haemolymph L-lactate concentrations were, in all but five instances over the trial,

below the detection threshold of the Accutrend™

portable lactate analyser.

Consequently, further investigation of this parameter is required using a more sensitive

assay method before any conclusion can be made regarding its viability as an indicator

of stress in L. polyphemus.

o All indicators point to adult L. polyphemus being capable of tolerating 2 hours of air

exposure (sufficient for bleeding purposes) with no long-term negative effects.

o Bacterial loading within the various components of the recirculating aquaculture system

(RAS) currently employed by Marine Biotech Ltd were deemed, at the time of

sampling, within tolerable limits.

o Of the 35 bacterial strains identified via the API®

20 E™

identification system, only four

could potentially behave pathogenically toward L. polyphemus. These are Aeromonas

hydrophila/caviae/sobria, Aeromonas salmonicida, Ewingella americana and Vibrio

fluvialis. Two further strains were identified as the potential human pathogens,

Pseudomonas oryzihabitans and Pasteurella multocida.

6. Recommendations

Improvements in husbandry practices

o The implementation of a carapace lesion monitoring system, via the use of a digital

camera, to allow comparison of wounds and lesions over time (i.e. during routine

examination or bleeding) as a means of assessing health status and identifying animals

that may require isolation and/or treatment.

o Improved management of the bacterial flora of the Marine Biotech Limited RAS system

through the relocation of the UV sterilisation unit from its current position to one

between the biofilter and the holding tanks, as well as its continual usage.

o Improvement of overall water quality and reduction of animal stress, either via an

increase in system volume (i.e. size of the biofilter and sump) or a reduction in system

biomass, as well as a more regimented diet and feeding regime.


23

o The introduction of protocols for hand washing after animal handling/contact with

culture water and the use of alcohol gel and gloves for staff suffering injuries with the

potential to become infected.

Further scientific studies

o Further assessment of haemolymph L-lactate concentration as a potential indicator of

stress in L. polyphemus.

o Investigation of the potential of metabolic profiling (metabolomics) as a means of

examining, in greater detail, the biochemical and physiological impact of stress on L.

polyphemus.

o Investigation into the cumulative effect/s of repeated air exposure and commercial

haemolymph extraction on the health status of captive L. polyphemus.

o Identification of an optimum recovery period post bleeding as well as the determination

of an optimum blood volume to mass ratio for commercial extraction.

Acknowledgements

This work was funded in part by a First Steps Award from the University Of Glasgow

Innovation Network. The access provided to holding facilities and tissue samples by

Marine Biotech Ltd. is gratefully acknowledged.

.


24

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31

Appendices

Appendix 1 Marine Biotech Limited Recirculating Aquaculture System (RAS) 32

Appendix 2 Bacterial colony counts (complete data) 34

Appendix 3 Isolate identification 36

Appendix 4 Final Report Form 45


32

Appendix 1

Marine Biotech Limited Recirculating Aquaculture System (RAS)

The system comprises two 3.8m diameter, circular, fibreglass holding tanks, protein

skimmer, sump with bag filters, UV sterilisation unit and a suspended bead column

biofilter. Water exits each holding tank via centrally located drain pipes, passes through the

protein skimmer then drains into the sump (via the numerous bag filters). After this the

water is pumped into the suspended bead column biofilter, either directly or via the UV

sterilisation unit. Water exits the biofilter through the return pipe which subsequently

divides to supply both holding tanks.

RAS employed at the Marine Biotech Limited facility near Callander, Scotland. Arrows

indicate direction of water flow. Photographs; a) and b) show the filtration system, c) the

two holding tanks, d) female animals in their holding tank.


33

Culture water is artificial seawater comprising Open Ocean™ mix dissolved in tap water

(left to settle for 48h with air stone prior to mixing). Total system volume is approximately

14,000 litres; each tank contains ca. 5700 litres, thereby leaving around 3,600 litres in the

pipes and filter system. 15% system volume (ca. 2000 litre) water exchanges occur every 4

- 8 weeks (dependant on ammonia levels).

Each holding tank contains ca. 100 adult L. polyphemus (segregated by sex). These are fed

a variable diet of sprat, sand eel, squid and mackerel (processed into segments 2-4cm in

size) at a rate of 750 – 1000 grams every 3 - 4 days. Abiotic culture conditions are as

follows:

• Temperature; 14 - 16°C

• Salinity; 25 - 30 ppt

• Dissolved Oxygen; minimum 70%

• pH; 7.8 - 8.6

• Ammonia and nitrate; low as possible

• Photoperiod; 11 hours (8am – 7pm)

Neutral buffered formalin (NBF) solution (10 %)

Formaldehyde (37-40%) 100 ml

NaH2PO 44.0 g

Na2HPO4 (anhydrous) 6.5 g

Distilled water 900 ml

Mix to dissolve.


34

Appendix 2

Bacterial colony counts (complete data)

Sample: Location: 24hrs: 48hrs: 1 Wk: 24hrs: 48hrs: 1 Wk: 24hrs: 48hrs: 1 Wk:

BB1 0 0 0 0 0 600 0 200 9200

BB2 0 0 0 0 120 840 0 80 2700

W4 0 0 0 2880 7120 9880 4320 5600 9160

W4* 0 400 400 3200 8400 12400 4800 9200 436400

W5 0 0 0 0 0 2120 0 0 3200

W5* 0 0 0 0 4400 9600 5600 8800 338400

W6 0 0 0 0 5120 7840 3320 6600 11400

W6* 0 0 0 0 5200 8400 4000 112000 163200

W7 40 40 40 1400 3240 4280 2280 5040 19400

W7* 0 0 0 1600 4800 8400 1600 6800 132400

W8 0 120 120 1480 3720 4520 160 2280 3320

W8* 0 400 400 0 7600 8800 2400 8800 11200

W9 0 0 0 1640 4560 5960 0 3560 7240

W9* 0 0 0 800 6800 11600 2400 6000 11600

W10 0 0 0 100 3560 5680 40 3960 7120

W10* 0 0 0 2000 3600 6400 1200 2400 3600

W11 40 200 200 840 3760 4920 1560 3360 5360

W11* 0 0 0 3200 4400 8400 0 4800 10000

W12 0 40 40 0 4480 6280 240 5160 9800

W12* 0 400 400 0 7600 12400 0 8000 55200

W13 0 160 160 800 5840 7280 1360 4160 6800

W13* 400 400 400 0 8400 12800 0 4800 10400

W14 40 200 200 0 3480 5720 480 640 5080

W14* 400 800 800 0 4400 9600 0 5600 12400

W15 0 120 120 1720 5720 8440 320 3920 9280

W15* 0 0 0 400 3200 6800 5200 4800 12400

‘Male’

Tank

Tank

Outflow

Protein

Skimmer

Outflow

Thiosulphate Citrate Bile Salts

Agar:Tryptone Soya Agar: Marine Agar:

Biofilter

Bead*

‘Female’

Tank

CFUs per ml derived from neat and 10 fold dilutions (*) of RAS water ('W') and biofilm bead ('B') samples

after 24 hours, 48 hours and 1 week of incubation at 15°C.


S1 0 0 0 0 720 62960 0 0 135360

S2 0 0 0 0 0 2000 0 0 16160

S3 0 0 0 0 0 80 0 0 0

S4 0 0 0 0 40 2200 0 0 1640

S5 0 0 0 0 40 2720 0 80 2720

S6 0 0 0 0 160 1800 0 0 2440

S7 0 0 0 0 120 1400 0 0 960

S8 0 0 0 0 120 2480 0 0 3920

S9 0 0 0 40 120 200 0 0 1240

S10 0 0 0 0 200 52320 0 80 50920

S11 0 0 0 0 0 7800 0 80 19760

S12 0 0 0 0 360 136900 0 80 85720

S13 40 40 40 0 0 840 0 0 1040

S14 0 0 0 0 0 3280 0 0 5120

S15 0 0 0 0 0 2360 0 0 2680

‘Female’

Tank

‘Male’

Tank

Sump

Tank

Biofilm

Bag Filter

Biofilm



Biofilter

Tank

Biofilm

CFUs per ml derived from RAS swab samples after 24 hours, 48 hours and 1 week of incubation at 15°C.


35


SC22A Carapace 0 0 0 0 0 3200 0 40 3320

SC22B Carapace 0 0 0 0 0 400 0 0 440

SC22C Carapace 0 0 0 0 80 600 0 0 440

SG22 Gill 0 0 0 0 0 0 0 0 600

SL22 Lesion 0 0 0 0 0 12960 0 1760 15360

SC33 Carapace 0 0 0 0 0 0 0 0 0

SG33 Gill 0 0 0 0 0 40 0 0 40

SC67 Carapace 0 0 0 0 0 0 0 0 200

SG67 Gill 0 0 0 0 0 0 0 0 0

SC104 Carapace 0 0 0 0 0 0 0 0 40

SG104 Gill 0 0 0 0 0 0 0 0 0

SC106 Carapace 0 0 0 0 0 0 0 0 0

SG106 Gill 0 0 0 0 0 0 0 0 0

SL106 Lesion 0 40 80 2360 4560 45520 2720 9160 77320

SC139 Carapace 0 0 0 0 0 0 0 0 0

SG139 Gill 0 0 0 0 0 0 0 0 0

SL183 Carapace 0 0 0 40 1720 52800 0 33040 68400



CFUs per ml derived from L. Polyphemus swab samples after 24 hours, 48 hours and 1 week of incubation at

15°C.

Sample: 24hrs: 48hrs: 1 Wk: 24hrs: 48hrs: 1 Wk: 24hrs: 48hrs: 1 Wk:

H22A 0 0 0 0 0 0 0 0 0

H22B 0 0 0 0 0 2 0 0 0

H33A 0 0 0 0 0 0 0 0 0

H33B 0 0 0 0 0 0 0 0 0

H67A 0 0 0 3 0 0 0 0 0

H67B 0 0 0 0 0 26 0 0 0

H104A 0 0 0 0 0 0 0 0 0

H104B 0 0 0 2 1 1 0 0 0

H106A 0 0 0 0 0 0 0 0 0

H106B 0 0 0 0 0 0 0 0 0

H139A 0 0 0 0 0 0 0 0 0

H139B 0 0 0 0 0 0 0 0 0



CFUs derived from haemolymph samples after 24 hours, 48 hours and 1 week of incubation at 15°C.


36

Appendix 3

Isolate identification

Isolate Sample Original Isolate Sample Original

Number: Number: Location: Number: Number: Location:

1 H22B Haemolymph (22) TSA 22 S14 Bag Filter TSA

2 H22B Haemolymph (22) TSA 23 S6 'Female' Tank TSA

3 H22B Haemolymph (22) TSA 24 S5 'Female' Tank MAR

4 W6* 'Female' Tank TSA 25 S9 'Male' Tank TSA

5 W6* 'Female' Tank TSA 26 S8 'Male' Tank TSA

6 W6* 'Female' Tank TSA 27 BB1 Biofilter Bead TSA



9 W6* 'Female' Tank MAR 30 BB1 Biofilter Bead TSA

10 W5* 'Female' Tank MAR 31 SC67 Carapace (67) MAR

11 W5 'Female' Tank MAR 32 SG33 Gill (33) MAR

12 W5 'Female' Tank MAR 33 SC106 Carapace (106) TCBS

13 W4* 'Female' Tank TCBS 34 SC106 Carapace (106) TCBS

14 W7* 'Male' Tank TSA 35 SL183 Lesion (183) MAR

15 W7* 'Male' Tank TSA 36 SL106 Lesion (106) MAR

16 W7 'Male' Tank TSA 37 SL106 Lesion (106) MAR

17 W11 Tank Outflow TCBS 38 SC22 Carapace (22) MAR

18 W8 'Male' tank TCBS 39 W6* 'Female' Tank MAR

19 W13 Protein Skimmer TCBS 40 SL22 Lesion (22) MAR

20 W14 Protein Skimmer TSA 41 S10 Sump Biofilm TSA

21 S15 Bag Filter TSA

Media Media

Isolates selected for API 20E identification, included; sample plate code, sample location and growth

media (TSA – red, TCBS – green, marine agar – blue).

1 Negative 12 Negative 22 Negative 32 Negative








11 Negative 21 Negative 31 Negative

Gram type: Isolate: Gram type:Gram type: Isolate: Gram type: Isolate:Isolate:

Gram type of isolates; growth media indicated by colour (TSA – red, TCBS – green, marine agar – blue).


37

Photographs of original sample plates from which isolates 1-34 were selected. Isolates 35-41 were taken from cross streaked plates (pictures not available). Note: the large white regions in the first image (isolates 1,

2, 3) is fungal growth.


38

1

Pasteurella

pneumotropica/

Mannheimia haemolytic

49.7Ochrobactrum

anthropi42.1

3 Ochrobactrum anthropi 60.7

Bordetella/

Alcaligenes/ Moraxella

spp

27.8

Pasteurella

pneumotropica/

Mannheimia

haemolytia

8.5

4Shewanella putrefaciens

group99.7

6Aeromonas hydrophila/

Caviae/Sobria 164.2 Vibrio fluvalis 31.4

Aeromonas

hydrophila/

Caviae/Sobria 2

24.2

7 Vibrio fluvialis 91.0

9 Non-fermenter spp. 67.0

Bordetella/


spp

10.6

Myroides spp/

Chryseobacterium

indologenes

10.4Pseudomonas

fluorescens/putida7.9

10Bordetella/ Alcaligenes/

Moraxella spp43.1 Non-fermenter spp. 30.1

Ochrobactrum

anthropi23.8

11

Pasteurella

pneumotropica/


49.7Ochrobactrum

anthropi42.1

12Pseudomonas

oryzihabitans88.1

Bordetella/Alcaligenes/


Ochrobactum

anthropi2.5

13Aeromonas hydrophillia/

Caviae/Sobria 199.2


group99.9

15 Citrobacter younge 98.6

16Bordetella/Alcaligenes/


Ochrobactrum

anthropi13.2

Pseudomonas



Aeromonas

hydrophillia/

Caviae/Sobria 1

18.1

Aeromonas

hydrophillia/

Caviae/Sobria 2

7.6

19Aeromonas salmonicidia

spp salmonicidia74.0 Burkholderia cepacia 11.7

Chromobacterium

violaceum4.2 Non-fermenter spp 4.1

Pseudomonas



Aeromonas

hydrophillia/

Caviae/Sobria 1

45.6

Aeromonas

hydrophillia/

Caviae/Sobria 2

6.5

21 Ochrobactrum anthropi 60.7Bordetella/Alcaligenes/

Moraxella spp27.8

Pasteurella

pneumotropica/

Mannheimia

haemolytia

8.5

Aeromonas hydrophila/

Caviae/Sobria 1

Strain Identity (Most

likely):

Strain Identity

(Least likely): %

8 55.8Aeromonas hydrophila/

Caviae/Sobria 223.5 Vibrio fluvialis 20.6

Strain Identity: % Strain Identity: %Isolate % Strain Identity: %


39

22 Non-fermenter spp. 67

Bordetella/


spp

10.6

Myroides spp/

Chryseobacterium

indologenes

10.4Pseudomonas


23 Ewingella americana 97.5 Burkholderia cepacia 1.7

24

Pasteurella

pneumotropica/


39.4Chyseobacterium

meningosepticum20.3 Burkholderia cepacia 18.9 Non-fermenter spp. 17.7

25 Burkholderia cepacia 94.9

26Bordetella/Alcaligenes/


Ochrobactrum

anthropi13.2

Pseudomonas

fluorescens/ putida3.6

27 Non-fermenter spp. 67

Bordetella/


spp

10.6

Myroides spp/

Chryseobacterium

indologenes

10.4Pseudomonas


29 Vibrio fluvialis 74.1Aeromonas hydrophila/

Caviae/Sobria 118.1

Aeromonas

hydrophila/

Caviae/Sobria 2

7.6


group99.7

31Bordetella/ Alcaligenes/


Ochrobactrum

anthropi23.8


Pasteurella

pneumotropica/

Mannheimia

haemolytia

17.9

Bordetella

/Alcaligenes/

Moraxella spp

13.3

Myroides spp/

Chryseobacturium

indolgenes

8.5Chryseobactrum

indologenes3.5

34 Grimontia hollisae 82.4 Pasteurella multocida 12.5


group99.9

36Aeromonas hydrophila/

Caviae/Sobria 182.7

Aeromonas hydrophila/

Caviae/sobria 217

37

Myroides spp/

Chryseobacterium

indologenes

34.1

Aeromonas

salmonicida spp

salmonicida

24.5Chryseobacterium

meningosepticum9.5

Chryseobacterium

indologenes6.6


Pasteurella

pneumotropica/

Mannheimia

haemolytia

17.9Bordetella/Alcaligenes/

Moraxella spp13.3

Myroides spp/

Chryseobacturium

indolgenes

8.5Chryseobactrum

indologenes3.5


group99.9

41 Ewingella americana 97.5 Burkholderia cepacia 1.7

Strain Identity (Most

likely):

Strain Identity

(Least likely): %Strain Identity: % Strain Identity: %Isolate % Strain Identity: %


40

Aeromonas salmonicida spp salmonicida - is a Gram-negative, non-motile bacterium (Herman,

1968). Causative agent of furunculosis in salmonids; a systematic disease responsible for significant

losses within the aquaculture industry (Lund et al, 1991; Lutwyche et al, 1995). Some vaccines are

available to prevent A. salmonicida outbreaks, however these are limited to organisms with specific

immune systems (Evenberg et al, 1988). Virulent strains of A. salmonicida possess A-protein; while

avirulent strains do not (Ishiguro et al, 1981). A. salmonicida was initially found within fish, and an

assumption was made that these bacterial cells are unable to survive outside of the host, however

Allen-Austin et al (1984) showed that the strain can survive in freshwater and cause disease

outbreaks when salmonids come in contact with contaminated water.

A. salmonicida has been found to cause mortality in Litopenaeus vannamei and can be harboured by

marine molluscs including the ebonyshell musssel, Fusconaia ebena (Buglione et al, 2010;

Starliper, 2005). Furthermore, the Aeromonas group is typically chitinoclastic (Janda, 1985). There

is no record of this strain detrimentally affecting L. polyphemus; however it is worth noting that one

of the two locations that this potential strain was found was within a carapace lesion on animal 106.

Given that this strain was also isolated from protein skimmer outflow water (isolate 019, 74%) it is

likely free living within the culture system given the lack of an active UV filter.

Bordetella spp., Alcaligenes spp. & Moraxella spp. - three genera of bacteria between which the

API® 20 E™ database is unable to distinguish. Each will be discussed separately in this section.

- Bordetella spp: small, Gram negative, highly fastidious, obligate aerobes (Doern, 2000). Some

strains are pathogenic to (largely immuno-compromised) humans, including B. holmesii (Guthrie et

al, 2010). Has been found in samples from a freshwater aquaculture facility near London, but has

not been associated with any aquatic disease outbreaks (Allen et al, 1983). However, Bordetella

bronchiseptica has been identified as a contributing factor towards the mass mortality in Caspian

seals, Phoca caspica, in 2000 indicating that some Bordetella species can be highly pathogenic

(Kuiken et al, 2006).

- Alcaligenes spp: Strains from this group have been found in a marine RAS containing a variety of

ornamental fishes (Raja et al, 2010). Some of the Alcaligenes strains are pathogenic to humans,

including Achromobacter (Alcaligenes) xylosoxidans which cause respiratory tract infections in

cystic fibrosis sufferers (Liu et al, 2002). Alcaligenes denitrificans was also isolated from the guts

of healthy L. vannamei (Franco et al, 2010.).

- Moraxella spp: a genus of bacteria usually isolated from seawater as well as seaweed and

decaying animals (Stolp, 1988). It has recently been isolated from various sampling sites in Mar

Piccolo of Taranto (Ionian Sea, Italy) that are important for the farming of Mytilus galloprovincialis

as well as on the mussels themselves, although no ill effects have been reported (Cavallo et al,

2009). Moraxella spp. have also been isolated from the hatchery samples of three sturgeon species,

but again with no pathogenicity observed (Masouleh et al, 2005).

Therefore, it is unlikely that Bordetella spp., Alcaligenes spp. & Moraxella spp. strains are likely to

present a problem to the HSCs or staff at Marine Biotech Limited.

Burkholderia cepacia complex - is a group of 17 closely related gram negative strains typically

found in water and soil (Leitao et al, 2010; Vial et al, 2011). Originally named Pseudomonas

capacia, but molecular analysis lead to the new genus of Burkholderia being created (Yabuuchi et

al, 1992). This group can be pathogenic to humans and causes pneumonia in immuno-

compromised individuals, especially those with pre-existing lung problems, particularly cystic

fibrosis (Martin et al, 2011; Leitao et al, 2010). Although the Burkholderia cepacia complex is

wide ranging, different strains predominate in different areas of the globe (Govan et al, 2007).

Caenorhabditis elegans, a nematode, is susceptible to pathogenic B. cepacia strains, with high

levels of mortality found (Cooper et al, 2009). The complex is also pathogenic to mice and rats and

some insects (Uhelinger et al, 2009). However, there appears to be no record of this strain affecting

aquaculture systems or marine arthropods. This strain is not thought to be of great concern to the

Marine Biotech Limited systems.


41

Chromobacterium violaceum - is a facultative anaerobic coccobacillus regularly found in water and

soil in the tropics and sub-tropics (Chang et al, 2007). C. violaceum is produces an antimicrobial

called Violacein (Hoshino, 2011). The strain rarely infects humans but on the rare occasion it does,

can prove fatal (Chang et al, 2007).

This strain was a possible identification for isolate 019; however, the likelihood was only 4.2%,

whilst the likelihood of this isolate being Aeromonas salmonicida was 74%. This combined with the

limited pathogenicity of the strain suggest that its potential occurrence within the Marine Biotech

Limited system is not of any concern.

Chryseobacterium indologenes - a non-fermentative, Gram negative bacillus; pathogenicity

towards humans is rare although it has been found in diabetic children (Cascio et al, 2005). The

Chryseobacterium group is ubiquitous in nature but is primarily found in soil and water (Kirby et al,

2004). C. indologenes is only rarely associated with infections in humans and appears to be mostly

associated with infections caught by already immuno-compromised patients within a hospital

environment or is transferred on internally implanted medical devices (Kirby et al, 2004).

There appears to be little or no information available on the occurrence of C. indologenes in marine

organisms or culture systems and the records of this strain involved with humans suggests that it

only causes problems in already immuno-compromised individuals and neonates, therefore it is

unlikely that this strain will cause any problems at the Marine Biotech Limited Facility.

Furthermore the likelihood of this strain being identified within the system is between 3.5%

(isolates 32 and 38) and 6.6% (isolate 37).

Chryseobacterium meningosepticum - has been reclassified as Elizabethkingia meningosepticum

(Kim et al, 2005). E. meningosepticum is found within infected wounds in humans as well as being

associated with skin ulcers on cod (Grawinski et al, 2009). It has also been isolated from uncooked

Ark shells, Scapharca broughtonii, (Yan et al, 2009). This strain is the most pathogenic of the

group and has been associated with neonatal meningitis and up to 57% mortality in this condition as

well as resulting in many long term post meningitis effects (Kirby et al, 2004).

This strain is obviously found within the marine environment and can be potentially pathogenic to

both a variety of marine organisms and humans alike. The level of pathogenicity is minimal in

adults, therefore should not cause any major problems to the animals or staff at Marine Biotech

Limited as long as numbers are kept relatively low.

Citrobacter youngae - was first described by Brenner et al in 1993. Citrobacter spp are facultative

anaerobic, motile Gram-negative, non-sporeforming bacillus (Borenshtein and Schauer, 2006;

Kurtoglu et al, 2011). They are members of the Enterobacteriaceae and utilise citrate as a carbon

source (Borenshtein and Schauer, 2006). C. youngae is considered pathogenic to humans and has

been isolated from human blood and stool samples as well as meat scraps (Brenner et al, 1993).

However, it is thought that immuno-competent adults are unlikely to develop Citrobacter infections

(Kurtoglu et al, 2011). C. youngae has been isolated from drinking water/river water samples,

whilst other species of Citrobacter have been isolated from the cloacal vent of green turtles,

Chelonia mydas, (Boualam et al, 2003; Al-Bahry et al, 2011). Yathavamoorthi et al (2010) found

Citrobacter species within the freshwater prawn, Macrobrachium rosenbergii, culture system in

India and thought that the high proportion of Citrobacter and Enterobacter species may have

significance to public health and indicate levels of water pollution. This strain is presumed

pathogenic, but could be useful as a marker of water quality in the Marine Biotech Limited RAS. It

can infect immuno-compromised humans but does not seem to be a major health threat to the L.

polyphemus or staff members at Marine Biotech Limited.

Ewingella Americana - is the only species of genus Ewingella (Grimont et al, 1983). E. americana

has low pathogenic potential but very occasionally infects immuno-compromised patients, causing

peritonitis (Pound et al, 2007). Most records of E. americana suggest that it is found within wounds

and sputum of patients already within hospitals (Bear et al, 1986), however, there is a record of it

possessing chitinolytic properties. This could result in the infection of L. polyphemus with wounds


42

or compromised carapaces (Inglis and Peberdy, 1997). Isolates 23 and 41 were potentially identified

as being E. americana, (97.5% likelihood).

Grimontia hollisae (formerly Vibrio hollisae) - is a member of family Vibrionaceae belonging to

genus Grimontia (Thompson et al, 2003).

This strain was isolated on TCBS agar, however, the literature states that G. hollisae is unable to

grow on TCBS agar (Thompson et al, 2003) indicating that this strain identification may be

incorrect. The API® 20 E™ database identified the strain as G. hollise with an 82.4% likelihood.

However, given the inability of this strain to grow TCBS media, isolate 34 is likely to be

Pasteurella multocida (12.5% likelihood).

Ochrobactrum anthropi - first described in 1988 by Holmes et al; and named anthropi as most

strains were isolated from human clinical specimens. O. anthropi are motile, using peritrichous

flagella form locomotion. The colonies are typically circular, low convex and smooth; a description

which fits with the appearance of isolates 03, 31 and 32, however, isolate 38 was a pale peach

colour, suggesting that 038 may not be O. anthropi.

This strain is a common component of marine biofilms (Lee et al, 2003) and has been found on the

spermatophores of the black tiger shrimp, Penaeus monodon (Nimrat et al, 2008). It is also an

infectious agent in Mekong giant catfish, Pangasianodon gigas and has been isolated from

nematodes in the Caribbean (Purivirojkul et al, 2005; Babic et al, 2000). Furthermore O. anthropi is

starting to be recognised as a pathogen of humans, with some infections responsible for life

threatening conditions such as endocarditis (Chain et al, 2011).

This strain should not necessarily be of concern to Marine Biotech Limited; however, it is important

to note that the potential of this strain as a pathogen has only recently emerged (Chain et al, 2011).

Myroides spp. – The Myroides genus contains various ‘aerobic, yellow-pigmented, non-motile, non-

fermenting, Gram-negative rods’ (Benedetti et al, 2011). They are widely distributed but are

particularly common in water, with strains such as Myroides marinus having been isolated from

seawater (Benedetti et al, 2011; Sung-Heun et al, 2011).

Myroides spp are potentially pathogenic, especially to immuno-compromised patients, however

infections have been seen in immunocompetent individuals (Benedetti et al, 2011). Infections in

animals appear to be rare or not yet investigated. Therefore there appears to be limited threat to the

animals and staff at Marine Biotech Limited.

Non-fermenter spp. - Non-fermenter species refers to any of a number of bacteria that are unable

to catabolise glucose and subsequently cannot ferment. The test results did not provide the API® 20

E™ database with sufficient data to provide a more accurate identification.

Pasteurella multocida - is a Gram negative coccobacillus and is a common oral bacterium of many

animals, including cats and dogs and is the primary cause of infection in animal bite wounds

(Weber et al, 1984). It can cause cellulitis, abscesses, osteomyelitis, septic arthritis and many other

conditions (Weber et al, 1984). This strain is also responsible for hemorrhagic septicaemia in cattle,

pneumonic pasteurellosis in sheep and goats, fowl cholera in poultry and atrophic rhinitis in pigs

and snuffles in rabbits (Ranjan et al, 2011). Additionally, P. multocida was found to be one of the

pathogenic strains responsible for a disease outbreak at the Laboratory of Marine Shrimp, UFSC,

Sant Catarina State, Brazil, and has been found to cause mortality in Litopenaeus vannamei

(Buglione et al, 2010).

Although it cannot be assumed that a shrimp pathogen would be harmful to L. polyphemus, the wide

spread pathogenicity of this strain (which includes mammals, birds and arthropods as well as

humans) makes it of concern. This strain is typically an opportunistic infectious agent in humans,

gaining entry via open wounds, suggesting that any staff at Marine Biotech Limited with injuries

should take precautions to prevent infection.


43

Pasteurella pneumotropica/Mannheimia haemolytica

- Pasteurella pneumotropica: is a short rod shaped bacterium; it is fairly ubiquitous and has been

isolated from the sediment of a fish pond in Saudia Arabia (Sasaki et al, 2011; Al-Harbi and Uddin,

2006). It is also a strain commonly found associated with upper respiratory tract infections

laboratory rats and mice(Sasak et al, 2011; Pritchett-Corning et al, 2009). The majority of

information regarding the pathogenicity of this strain is focused on rodents. However, there appears

to be very little, if any, threat to other animal groups. It is felt that this strain should not negatively

impact L.polyphemus.

- Mannheimia haemolytica: is Gram negative bacterium found associated with infections of the

upper respiratory tract of ruminants (Frank, 1989). It is responsible for a number of economically

important cattle diseases (van Rensburgh et al, 2004). There appears to be no record of this strain

within aquaculture or non-ruminant species.

P. pneumontropica and M. Haemolytica appear to be limited in their pathogenicity to rodents and

ruminants, respectively. The highest likelihood of an isolate being either of these strains is 49.7%

(isolates 01 and 11). Isolate 01 was obtained from the haemolymph of crab 22 and is likely the

result of environmental contamination. Also given the relatively low I.D. accuracy and the strains’

exclusivity to rodents and ruminants suggests that this may be an incorrect identification.

Pseudomonas fluorescens/putida - both species are Gram negative and rod-shaped, with P.

fluorescens possessing multiple flagella (Klinge, 1959). Both of these strains are commonly isolated

from soil samples (Cornelis, 2010). However Borges et al (2008) found P. fluorescens within the

biofilter of a recirculating aquaculture system. The highest likelihood for these strains being the

correct identification for any of the isolates was 7.9% (isolates 09, 22 and 27). However, it is not

thought that these strains would present a problem to L. polyphemus or staff at Marine Biotech

Limited.

Pseudomonas oryzihabitans - is a non-fementative, Gram negative bacterium, previously found

associated with catheter-related infections in humans (Esteban et al, 2002). P. oryzihabitans was

also found to be the causative agent in an ulcerous lesion on the forearm of a young boy resulting

from a bite from an Octopus vulgaris (Aigner et al, 2011).The strain was determined to be an 88.1%

match for isolate 12, a water sample from the ‘female’ holding tank. Given the resultant infection

from the O. vulgaris bite it is recommended that staff at the Marine Biotech Limited facility take

care to avoid allowing culture water to contact broken skin or mucus membranes (such as the mouth

or eyes).

Shewanella putrefaciens group – S. putrefaciens is a facultative anaerobe previously isolated from

marine environments and is thought to play an important role in the cycling of sulphur and trace

metals in marine and freshwater habitats (Perry et al, 1993). The name ‘putrefaciens’ is derived

from the bacteria’s association with purification. S. putrefaciens was found to be the dominant

strain on Penaeus vannamei during cold storage (Zhao et al, 2011) and is considered to be a specific

spoilage organism of marine cold-water fish during ice storage (Tryfinopoulou et al, 2007). The

I.D. accuracy for S. putrefaciens was 99%, providing strong evidence to support that this strain is a

component the RAS microflora. Two isolates (35 and 40) identified as S. putrefaciens were

collected from carapace lesions on individuals 183 (female) and 22 (male). It is potentially of

concern that S. putrefaciens, a bacteria primarily associated with putrefaction in marine organisms,

was found within the lesions of at least two animals at Marine Biotech Ltd.

Vibrio fluvialis - Vibrios are ubiquitous to the marine environment with a corresponding proportion

being pathogenic to marine organisms (Tall et al, 2003). V. fluvialis is a halophillic, slightly curved,

rod-shaped, Gram negative bacterium. It has been identified in samples of seawater, animal faeces,

human faeces, sewage and seafood products (Igbinosa and Okoh, 2010).

V. fluvialis causes cholera-like diarrhoea in humans and is resistant to many antimicrobial agents

(Chowdhury et al, 2011). However, records of it pathogenicity in non-human hosts are limited (Tall

et al, 2003). V. Fluvialis has been isolated from Hawaiian green turtles (Chelonia mydas) suffering

from fibropapilloatosis, however, it was only found in 47% of animals tested suggested that it was

not the causative agent (Aguirre et al, 1994). Tall et al (2003) implicated V. Fluvialis -like strains in


44

limp lobster disease; which causes lethargy, weakness and unresponsiveness to stimuli. Like most

of the Vibrionaceae, V. fluvialis has been demonstrated to be chitinolytic (Osawa ann Koga, 2008).

None of the isolates identified as V. fluvialis were directly associated with L. polyphemus. However,

due to its chitinolytic properties and pathogenicity this strain should be viewed as a potential health

threat to L. polyphemus. More significantly, this strain is known to cause cholera-like symptoms in

humans and therefore could pose a health risk to staff at Marine Biotech Limited.


45

Appendix 4

Final Report Form

University Of Glasgow Innovation Network

First Step Award with Marine Biotech Ltd.

Academic Partner

Professor Douglas M. Neil

Research Institute of Biodiversity, Animal Health and Comparative Medicine,

College of Medical, Veterinary & Life Sciences,

Room 312, Graham Kerr Building,

University of Glasgow,

Glasgow, G12 8QQ,

Scotland, UK.

Tel: ++44 (0)141 330 5969

Fax: ++44 (0)141 330 5971

Email: [email protected]

SME Partner

Alex Mühlhölzl

Managing Director

Marine Biotech Limited

Mobile: +44 (0) 787 613 6446

Email: [email protected]

PART ONE

(This section of the report will be made available for publication on the University of

Glasgow website and for use by Scottish Enterprise and The Scottish Government.)

Project Description – max. 500 words – please state whether the original project

description goals have been achieved, describe the tangible outcomes, commercial

opportunities, and any unforeseen benefits.

The project assessed the effects of a simulated commercial bleeding procedure on several

physiological indicators of stress in the American horseshoe crab, Limulus polyphemus. The main

Indicators utilised were: total haemocyte count (THC), concentrations of haemolymph L-lactate and

protein, blood pH and blood temperature. In addition, the bacterial loading and composition of the

re-circulating aquaculture system and animal microflora were examined for the presence of

potentially pathogenic strains, using both traditional culture methods and the Biomerieux API® 20

E™ bacterial testing system. The main findings obtained were:

o Haemolymph pH appears to provide an effective and practical physiological indicator for

stress induced by air exposure in adult L. polyphemus.

o Adult L. polyphemus appear capable of tolerating at least 2 hours of air exposure in

association with a bleeding procedure, with no long-term negative effects.


46

o Bacterial loading within the various components of the recirculating aquaculture system

(RAS) currently employed by Marine Biotech Limited was, at the time of sampling, within

acceptable limits.

o In total, 35 bacterial strains were identified via the API® 20 E™ testing system, and of

these four were potentially pathogenic to L. polyphemus (Aeromonas hydrophila/

caviae/sobria, Aeromonas salmonicida, Ewingella americana and Vibrio fluvialis). In

addition, two further strains were identified as the potential human pathogens,

Pseudomonas oryzihabitans and Pasteurella multocida.

The project achieved all its original goals. Regarding tangible outcomes, measures have been

identified to improve the holding conditions and to optimise husbandry practices for the culture of

L. polyphemus, at Marine Biotech Ltd’s facility. Implementation of these measures would reduce

the likelihood of stock loss through environmental stress, bleeding procedures and disease. The

main recommendations are:

o Increase control over the bacterial flora in the re-circulating seawater through modification

of the recirculating aquaculture system. This could be achieved via the relocation of the UV

filter unit from its current position to a position between the biofilter and holding tanks, as

well as bringing it into continuous usage. In addition, an increase in biofilter and sump

volume, as well as the implementation of a more regimented feeding regime, would also

improve overall water quality and consequently reduce animal stress.

o Implement a carapace lesion monitoring system, via the use of digital camera images, to

allow comparison of wounds and lesions over time (i.e. during routine

examinations/bleeding). Such a system would be a useful indicator of health status and

would aid greatly in the identification of animals requiring isolation and treatment.

o Investigate the cumulative effect(s) of repeated air exposure and haemolymph harvesting on

the health status of L. polyphemus, as well as the determination of an optimum recovery

period post-bleeding.

o Investigate the potential of metabolic profiling (metabolomics) as a means of determining

in more detail the biochemical and physiological impact of bleeding stress on L.

polyphemus.

Through implementation of the above recommendations the company could improve the holding

conditions for brood stock, thereby reducing the occurrence of disease and mortality. Healthier

brood stock typically provides better quality gametes, and these in turn increase the likelihood of

the production of viable larvae. Such improvements in production would increase operational

efficiency, and may also lead to new commercial opportunities for Marine Biotech Ltd.

PART TWO

(This section of the report is confidential and will not be made available for publication,

and will only be made available to project partners.)

SME Contribution – Please detail the contribution made by the SME to the project.

The SME management aided in defining the remit of the project and provided information on areas

of concern regarding animal holding conditions, through regular meetings with the scientific team.

The SME provided access to their brood stock for experimental purposes, as well as facilities for

sampling and limited consumables.


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Future Plans – do you plan to take this work forward? Please describe plans for development(s)

evolving from this project, has there been any intellectual property generated (or likely to be), have

you applied for (or plan to apply for) any research grants to develop this work further? The academic partner will honour the confidentiality agreement covering the work performed, and

will seek permission from the company to present and publish the scientific outcomes of the work,

where appropriate.

There is scope to extend this project to investigate further the responses of L. polyphemus to

commercial bleeding, using metabolic profiling techniques. Such metabolomic analysis would be

extremely useful in the determination of an optimum post-bleed recovery protocol.

In addition, the long-term goal of Marine Biotech Limited is the creation of a commercial scale L.

polyphemus nursery facility. There is a great deal of research potential associated with such an

endeavour as larval development has strict requirements with regard to environmental conditions

and diet.

Funding for these possible future developments might be sought from KTP, SMART or SPARK

awards, or other appropriate industry/science initiatives.

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